The structure of the GPR158 homodimer in
complex with RGS7-Gb5 shows that one RGS7-
Gb5 heterodimer interacts with two sites on
GPR158 (Fig. 1B and fig. S4B). The first in-
terface is a two-helical bundle comprising a
C-terminal helix (CT-CC) from each protomer.
Thesecondcontactsurfaceisatthedimer
interface between the seven-transmembrane
(7TM) regions of the GPR158 protomers. We
found that in the RGS7-bound state, the den-
sity for the GPR158 ectodomain becomes dif-
fuse. Although the density is observable at low
contour, the model could not be built. This
higher conformational flexibility at the ecto-
domain, possibly induced by RGS7-Gb5 bind-
ing, suggests that extracellular and intracellular
elements of the complex could be allosterically
connected. This notion is further supported
by a three-dimensional variability analysis,
which showed that ECD undulations coin-
cide with RGS7-Gb5 binding (movies S1 and
S2). Along with that, the GPR158 CT-CC andthe RGS domain of RGS7 show a higherB
factor, reflecting conformational dynamics
at these regions (fig. S4D). Cross-linking mass
spectroscopy (XL-MS) confirmed the main con-
tact points in the complex at the single amino
acid level (fig. S5).
Detailed analysis of the 7TM domain of
GPR158 reveals a compact dimeric interface
(Fig. 2A and fig. S6A). Superposition of the
two protomer 7TM regions shows a similar
arrangement of all the elements including
extra- and intracellular loops with RMSD value
of 0.35 Å (fig. S6B). Within each protomer,
ECL2 Cys^573 forms a disulfide bond with TM3
Cys^481 , an interaction conserved through-
out GPCRs. Thebhairpin of ECL2 interacts
with the stalk-TM linker through hydropho-
bic interactions, bridging the ectodomain
and TM domain of each protomer. ECL2 also
interacts with ECL1 and TM3 hydrophobi-
cally and with ECL3 through polar interac-
tions (Fig. 2B).
The dimer interface at the 7TM domain can
be separated into two parts. The first is formed
at the extracellular end by TM4, TM5, and
ECL2 of each protomer. The helices assemble
in an inverted V-shape, creating a cavity. The
second interaction at the cytoplasmic end
closes the cavity (Fig. 2C and fig. S6C). This
type of dimeric architecture has not been seen
in other GPCRs, including class C receptors
(fig. S6D).
The extracellular portion of the TM inter-
face formed by TM4, TM5, and ECL2 features
a series of hydrophilic interactions and hydro-
phobic contacts (Fig. 2C). The cytoplasmic
portion of the TM interface is formed by TM3
and ICL2 of both the protomers. The ICL2
regions interact with each other, and resi-
dues in TM3 form a basic patch that engages
phospholipids (fig. S6, C and E). The polar
contacts between Gln^516 across protomers also
form the lid that covers the cavity from the
intracellular end (II in Fig. 2C). The cavity
formed at the dimer interface is further shielded
at both its intra- and extracellular ends by
several cholesterol molecules from both the
front and back sides (fig. S6A). The TM do-
main interaction with cholesterols may sta-
bilize the dimeric interface.
We observed densities for two phospholip-
ids at the cavity formed by the TM dimeric
interface and performed mass spectrometry
to identify lipids copurified with the GPR158
complex (fig. S6F). Although not exhaustive,
this lipidomics analysis identified several
phospholipid species, most notably phos-
phatidylethanolamine (PE) and phospha-
tidylinositol (PI). Both molecules are well
accommodated into the respective cryo-EM
densities (Fig. 2A and fig. S3). The phospho-
lipids are arranged in the cavity in such a
way that one hydrophobic tail of each lipid is
inserted into the shallow hydrophobic pocketSCIENCEscience.org 7 JANUARY 2022¥VOL 375 ISSUE 6576 87
Fig. 1. Cryo-EM structures of GPR158 in its apo and RGS7-Gb 5 Ðbound states.(A) Cryo-EM map (left)
and model (right) of GPR158 homodimer in ribbon representation with protomers colored in cyan and pink.
Phospholipids PE and PI are shown in green and blue, respectively; cholesterols are yellow. (B) Cryo-EM
map (left) and model (right) of GPR158 homodimer complexed with RGS7-Gb5 in ribbon representation
colored as in (A); RGS7 and Gb5 are shown in olive and brown.
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